Gene silencing is essential for normal development and protection of the genome from selfish genetic elements, such as retroposons. Perturbation of normal gene silencing results in genomic instability, impaired response to DNA damage, progression from normal to premalignant states, and eventually to cancer. While progress has been made in understanding silencing mechanisms in condensed heterochromatin, the silencing of genes in open euchromatin is less well understood. For example, recruitment of histone deacetylases (HDACs) to heterochromatic regions is essential for maintenance of the silent state. However, we have found that the mouse Mixed-Lineage Leukemia 5 (MLL5) protein and its Drosophila ortholog, UpSET, recruit HDACs to active genes to modulate open chromatin, fine-tune gene expression, and prevent inappropriate activation of silent neighboring genes. In addition, while histone turnover and the histone deposition machinery are essential for gene expression in differentiated cells, using novel methodology developed in our lab, we found that they are also key players in establishing gene silencing in embryonic stem cells (ESCs). Our proposed experiments use genetic, developmental, cell biology, biochemical, and molecular approaches in complementary systems (mice and flies) to determine the mechanisms underlying these novel regulatory pathways and elucidate their roles in hematopoiesis and stem cell biology. Specifically, we propose to: 1. Define the mechanisms by which MLL5/UpSET containing complexes modulate transcription. Using our novel protein tethering method we will determine the transcriptional role of mammalian MLL5 containing complexes in hematopoiesis. Taking advantage of the functional and structural conservation of the Drosophila ortholog of MLL5, UpSET, we will define how recruitment of these proteins modulates transcribed chromatin architecture around promoter regions to precisely orchestrate gene expression. 2. Elucidate the functional role of MLL5 in DNA damage response and repair. Like other proteins involved in gene silencing, MLL5 also participates in DNA damage repair. We will elucidate the role of MLL5 complexes in the DNA damage response and determine how mutation of Mll5 alters this response. We will also determine if lack of MLL5 increases tumor incidence or latency in tumor-sensitive backgrounds. Such results would provide the molecular basis for the DNA damage sensitivity of Mll5 null mice and association of poor prognosis preleukemic syndromes with haploinsufficiency of chromosome band 7q22 where human MLL5 resides. 3. Discover the molecular mechanisms by which SRCAP mediates silencing in ESCs and how perturbation of this silencing impacts hematopoiesis. We will elucidate the molecular basis of a previously unknown ESC silencing mechanism we recently discovered involving the Snf2-related CREBBP activator protein (SRCAP) H2A.Z deposition complex and polycomb repressive complex (PRC)1. We will determine how H2A.Z deposition by SRCAP underlies PRC1-mediated silencing and shapes the ESC and hematopoietic transcriptional landscapes.